Ykl-40 monoclonal authority

ABSTRACT

The present invention relates to monoclonal anti-human YKL-antibodies which are capable to modulate biological processes in which YKL-40 plays a prominent role, e.g. inhibit the growth and/or inducing apoptosis of cells, in particular cancer cells. The invention also relates to pharmaceutical compositions comprising said antibodies and uses said antibodies and/or pharmaceutical compositions for treatment of a disease wherein inhibition of cell growth, cell differentiation, remodelling of extracellular matrix, metastasis and/or induction of cell death due to apoptosis is a prerequisite for successful curing. An antibody of the invention is capable of inhibiting biological function of YKL-40 in the above mentioned processes by binding to a specific epitope on YKL-40.

FIELD OF INVENTION

The present invention relates to monoclonal anti-human YKL-40antibodies, which are capable to inhibit the growth and/or inducingapoptosis of cells, in particular cancer cells, pharmaceuticalcompositions comprising said antibodies and uses said antibodies and/orpharmaceutical compositions for treatment of a disease whereininhibition of cell growth, cell differentiation, remodelling ofextracellular matrix, metastasis and/or induction of cell death due toapoptosis is a prerequisite for successful curing.

BACKGROUND OF INVENTION

YKL-40 was first identified as a protein secreted in large amounts by ahuman osteosarcoma cell line MG63 in vitro and was named according tothe last three N-terminal amino acids of its amino acid sequence, Tyr(Y), Lys (K) and Leu (L), and approximate molecular weight of 40 kDa(Johansen et al., 1992).

Analysis of the amino acid sequence of YKL-40 revealed that the proteinbelongs to the glycosyl hydrolase family 18 (Hakala et al., 1993). Thefamily consists of enzymes including chitinases and also proteins, whichdo not have enzymatic activity. The gene for YKL-40 (CHI3L1) is locatedon chromosome 1q31-1q32 and consists of 10 exons and spans about 8kilobases of genomic DNA (Rehli et al., 1997). The Sp1-familytranscription factors seem to have a predominantly role in controllingYKL-40 promoter activity (Rehi et al., 2003). The crystallographicstructure of human YKL-40 has been described (Fusetti et al., 2003;Houston et al., 2003). The protein contains two globular domains: a bigcore domain which consists of a (β/α)₈ domain structure with atriose-phosphate isomerase (TIM) barrel fold, and a small α/β domain,composed of five antiparallel β-strands and one α-helix, inserted in theloop between strand β7 and helix α7. This confers to the active site ofYKL-40 a groove-like character. The structure of YKL-40 is in partsimilar to the structure of several proteins, e.g. human chitotriosidase(Fusetti et al., 2002), mouse Ym1 (Sun et al., 2001), Drosophilamelanogaster imaginal disc growth factor-2 (Varela et al., 2002) andsome other members of the glycosyl hydrolase family 18 (Coulson, 1994),but there are also major differences. One of these differences is amutation of one of the three amino acids (Asp, Glu and Asp) essentialfor the chitinase-like catalytic activity, namely Glu->Leu, whichcompletely rules out the function of YKL-40 as a glycolytic enzyme.YKL-40 can binds chitin, but has no enzymatic activity (Renkema et al.,1998). YKL-40 is a glycoprotein and this is also a unique feature ofYKL-40 comparing to the other mammalian chitinase-like proteins.

YKL-40 can bind heparin. The amino acid sequence of the protein containsone heparin binding motif (¹⁴³GRRDKQH¹⁴⁹) which is located in a surfaceloop (Fusetti et al., 2003). YKL-40 can also bind hyaluronan. The foldedprotein contains two potential hyaluronan binding sites on the externalface. Human YKL-40 can bind chitin of different lengths in a similarfashion as chitinases of the Family 18. Nine sugar-binding subsites wereidentified in the 43 Å groove of YKL-40 (Fusetti et al., 2003). Bindingof short or long oligosaccharides to human YKL-40 is also possible. Thepresence of two distinct binding sites with selective affinity for longand short oligosaccharides (not found in other mammalian chitinase-likeproteins) may have significance for the functional activity of YKL-40 asa cross-linker between two targets carrying these oligosaccharides.

YKL-40 possesses a number of biological activities. Thus, it has beenshown that human YKL-40 can act as a growth factor for cells ofconnective tissue, such as chondrocytes and synovial cells (De Ceunincket al., 2001; Recklies et al., 2002). YKL-40 also promotes the growth offibroblasts in a fashion similar to insulin-like growth factor 1 (IGF-1)(Recklies et al., 2002). In fibroblasts YKL-40 initiates activation ofMAP kinase and PI3K signalling cascades leading to phosphorylation ofboth the extracellular signal-regulated kinase (ERK)-1/2 MAP kinase andprotein kinase B (AKT). Activation of cytoplasmic signal-transductionpathways by YKL-40 has been suggested to be mediated through interactionof YKL-40 with one or more receptor molecules on the plasma membrane,but the identity of the cellular receptor mediating the biologicaleffects of YKL-40 is currently unknown.

It has also been demonstrated that YKL-40 acts as a chemoattractant forendothelial cells and stimulates migration of these cells comparable tostimulation by basic fibroblast growth factor (Malinda et al., 1999).

YKL-40 modulates vascular endothelial cell morphology by promoting theformation of branching tubules, indicating that YKL-40 may play a rolein angiogenesis by stimulating the migration and reorganization ofvascular endothelial cells (Malinda et al., 1999). YKL-40 is also anadhesion and migration factor for vascular smooth muscle cells(Nishikawa et al., 2003). It has been suggested that YKL-40 may affectthe local extracellular hyaluronan concentrations available for cellattachment via two independent mechanisms: by binding to theextracellular hyaluronan, and by interfering with the synthesis andsecretion of hyaluronan by cells. Thus, YKL-40 may influence the extentof cell adhesion and migration during the tissue remodeling processesthat take place during inflammation, fibrosis, atherogenesis and cancergrowth and cancer metastasis.

In mice YKL-40 was called the “breast regression protein (Brp-39)”because the expression of the protein was induced in mammary epithelialcells a few days after weaning. It was proposed (Mohanty et al., 2003)that YKL-40 is involved in regulation of programmed cell death duringmammary involution as a protective signalling factor that determineswhich cells are to survive the drastic tissue remodelling that occursduring involution.

YKL-40 is expressed by different types of cells in vitro and in vivo, inparticular in tissues characterised by inflammation,degradation/remodeling of the extracellular matrix or ongoingfibrogenesis. YKL-40 is secreted by activated neutrophils (Volck et al.,1998), by macrophages during late state of differentiation (Kirckpatricket al., 1997; Krause et al. 1996; Rehli et al., 1997; Renkema et al.,1998; Rehli et al., 2003), arthritic chondrocytes (Hakala et al., 1993;Johansen et al. 2001; Volck et al. 2001), differentiated vascular smoothmuscle cells (Shackelton et al., 1995; Malinda et al., 1999; Nishikawaet al. 2003) and fibroblast-like synovial cells (Hakala et al. 1993;Nyirkos et al., 1990; Dasuri et al., 2004). Studies in human fetalchondrocytes indicate that YKL-40 is a differentiation marker(Imabayashi et al., 2003). In vivo YKL-40 mRNA and proteins expressionare found by a subpopulation of macrophages in inflamed synovialmembrane (Kirkpatrick et al., 1997; Baeten et al., 2000; Volck et al.,2001), atheromatous plaques (Boot et al. 1999), arteritic vessels frompatients with giant cell arteritis (Johansen et al., 1999a) and byarthritic chondrocytes (Volck et al., 2001), and peritumoral macrophagesin biopsies from small cell lung cancer express YKL-40 mRNA (Junker etal., 2005b).

A strong expression of YKL-40 mRNA in human liver has been shown to beassociated with the presence of fibrosis. Immunohistochemical studies ofliver biopsies have shown YKL-40 protein expression in areas of theliver with fibrosis, whereas no expression was observed in hepatocytes(Johansen et al., 1997; Johansen et al. 2000). Suppression subtractivehybridization analysis and RT-PCR have demonstrated that YKL-40 is oneof the most overexpressed proteins in cirrhotic liver tissue caused byhepatitis C virus (HCV) (Shackel et al., 2003).

Patients with non-malignant diseases characterized by inflammation andfibrosis such as active rheumatoid arthritis (Johansen et al., 1993;Harvey et al., 1998; Johansen et al., 1999b; Volck et al., 2001), severebacterial infections (Nordenbaek et al., 1999; Kronborg et al., 2002),active inflammatory bowel disease (Koutroubakis et al., 2003; Vind etal., 2003), and liver fibrosis (Johansen et al., 1997; Johansen et al.,2000; Tran et al., 2000; Nøjgaard et al., 2003) have elevated serumYKL-40.

YKL-40 is expressed and secreted by several types of human carcinoma(breast, colon, lung, kidney, ovarian, prostate, uterine, osteosarcoma,oligodendroglioma, glioblastoma and germ cell tumors) (A search of theYKL-40 sequence against the dbest database at the National Center forBiotechnology Information; Johansen et al., 1992; Junker et al., 2005a),and by murine mammary tumors initiated by neu/ras oncogenes (Morrison etal., 1994). Microarray gene analyses have identified the YKL-40 gene asone of the most overexpressed genes in papillary thyroid carcinoma(Huang et al., 2001), high-grade malignant gliomas (Tanwar et al.,2002), and extracellular myxoid chondrosarcoma (Sjogren et al., 2003).YKL-40 is expressed and secreted in vitro by the osteosarcoma cell lineMG63, glioblastoma cells and myeloid leukemia cell lines (U937, THP-1,HL-60) (Johansen et al., 1992; Rehli et al., 2003; Kirkpatrick et al.,1995; Verhoeckx et al., 2004). YKL-40 is not expressed by small celllung cancer cell lines in vitro nor in vivo but strongly expressed bytumor associated macrophages in small cell lung cancer biopsies (Junkeret al., 2005b).

A number of studies has now reported an elevated level of YKL-40 proteinin serum of cancer patients (Johansen et al., 1995; Cintin et al., 1999;Cintin et al., 2002; Tanwar et al., 2002; Brasso et al., 2003; Dehn etal., 2003; Geertsen et al., 2003; Høgdall et al., 2003; Jensen et al.,2003; Johansen et al., 2003; Dupont et al., 2004; Johansen et al.,2004). Several studies have demonstrated that an elevated serumconcentration of YKL-40 in patients with breast-, colorectal-, ovarian-,kidney-, small cell lung-, and prostate carcinomas is an independentprognostic parameter of short recurrence free interval and short overallsurvival. This observation has been done in patients with local oradvanced cancer at time of first cancer diagnosis and at time of relapse(Johansen et al., 1995; Cintin et al., 1999; Cintin et al., 2002; Brassoet al., 2003; Dehn et al., 2003; Geertsen et al., 2003; Høgdall et al.,2003; Jensen et al., 2003; Johansen et al., 2003; Dupont et al., 2004;Johansen et al., 2004). Based on these and other findings YKL-40 wassuggested as a diagnostic marker of the presence or absence of a cancerand for the prognosis of cancer recurrence and survival of cancerpatients (WO 00/19206), and it was described as a marker for degradationof connective tissue and used in methods for identifying the presence ofa disease associated with degradation of connective tissue (e.g. cancer)described (WO 95/01995; U.S. Pat. No. 5,935,798). Both groups of lattermethods are based on employing an anti-YKL-40 antibody for detecting theprotein in samples from the patients.

Antibodies against YKL-40 has long been known in the art and used forexample for the detection and monitoring the level of YKL-40 in bloodserum/plasma of cancer patients, however, functional anti-YKL-40antibodies, which would be capable of inhibiting the function of YKL-40,have not been produced nor described.

REFERENCES

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SUMMARY OF INVENTION

The present invention relates to a finding that monoclonal antibodiesagainst human YKL-40 are able to significantly inhibit cell growth, celldifferentiation, cell survival and metastasis.

Thus, in one aspect the present invention relates to a monoclonalanti-human YKL-40 antibody, antigen binding fragment or recombinantprotein thereof, said antibody, binding fragment or recombinant proteinthereof being capable of

-   -   inhibiting cell growth, such as e.g. endothelial, fibroblast,        synoviocytes, chondrocyte, hepatic stellate cells, vascular        smooth muscle cells, monocyte/macrophage or cancer cell growth,        and/or    -   inducing cell death, such as e.g. endothelial, fibroblast,        synoviocytes, chondrocyte, hepatic stellate cells, vascular        smooth muscle cells, monocyte/macrophage or cancer cell death,        and/or    -   inhibiting cell differentiation, such as e.g. endothelial,        fibroblast, synoviocytes, chondrocyte, hepatic stellate cells,        vascular smooth muscle cell, monocyte/macrophage or cancer cell        differentiation, and/or    -   inhibiting cancer cell metastatic potential.

A monoclonal anti-human YKL-40 antibody according to the invention iscapable to inhibit functional activity of human YKL-40 associated withcell growth, differentiation, survival and extracellular tissueremodeling. In particular, the invention relates to an antibody which iscapable of inhibiting biological activity of YKL-40 in

-   -   development/progression of cancer cell growth, and/or    -   development/progression of cancer cell metastasis, and/or    -   development/progression of cancer cell differentiation, and/or    -   development/progression of cancer cell survival, and/or    -   development/progression of angiogenesis, and/or    -   development/progression of extracellular matrix remodeling,        and/or    -   development/progression of liver fibrosis, and/or    -   development/progression of lung fibrosis, and/or    -   development/progression of tissue fibrosis, and/or    -   development/progression of organ fibrosis, and/or    -   development/progression of rheumatoid arthritis.

The invention features an antibody which is capable of inhibitingbiological function of YKL-40 in the above mentioned processes uponbinding to a specific epitope on YKL-40.

The invention, thus, in further aspects relates to an epitoperecognizable by the above antibodies. The epitope of the invention ischaracterised in that it comprises amino acid residues of any of thesequences identified herein as SEQ ID NOs: 2, 3, 4, 5, 6, or 7, orconsists of at least one of said sequences or a fragment thereof.According to the invention said epitope is a binding site, constitutes apart of a binding site of YKL-40 to its receptor, or is involved inassisting the activation of the receptor by YKL-40 by interacting withsome other molecules, for example an YKL-40 ligand, such as for exampleheparin, heparan sulfate proteoglycans, or hyaluronan.

Use of an antibody capable of recognizing the epitope as defined abovefor the

i) inhibiting cell growth (including cancer) and/orii) inhibiting cell differentiation (including cancer) and/oriii) inhibiting cell survival (including cancer) and/oriv) inducing cell death (including cancer) and/orv) inhibiting development/progression of extracellular matrixremodeling, and/orvi) inhibiting development/progression of liver fibrosis, and/orvii) inhibiting development/progression of rheumatoid arthritis, and/orviii) inhibiting development/progression of organ fibrosis, and/orix) inhibiting development/progression of tissue fibrosis, and/orx) inhibiting development/progression of angiogenesis, and/orxi) inhibiting metastasisis also an aspect of the invention. The invention in particular featuresuses of the antibodies for the treatment of cancer and/or non-malignantdiseases, such as for example inflammatory diseases and diseasesassociated with fibrosis.

The invention also relates to a pharmaceutical composition comprisingthe above antibody, antigen binding fragment or recombinant proteinthereof.

The invention also relates to peptide sequences comprising or beingcomprised by the epitope for the

-   -   i) production of an antibody of the invention, and/or    -   ii) modulating functional activity of YKL-40 receptor associated        with cell proliferation, differentiation and survival, and/or    -   iii) modulating biological functions of YKL-40, and/or    -   iv) manufacture of a medicament.

The invention concerns pharmaceutical compositions comprising theantibodies and/or peptide sequences as defined above as well.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the effect of different monoclonal antibodies against humanYKL-40 (mAb 115F9, 116F9 and 201F9) on cell growth and survival ofmalignant human glioblastoma U87 cells in vitro (MTT assay). Results aregiven as the mean value of 6 measurements+/−standard deviation.

FIG. 2 demonstrates the inhibiting effect of mAb 116F9 on cell growthand survival of human osteosarcoma cells U2OS and MG-63 and humanmalignant melanoma cells SK-MEL-28 in vitro (MTT assay). Results aregiven as the mean value of 6 measurements+/−standard deviation.

FIG. 3 shows the Kaplan-Meier plots of the growth of individual tumors(days until tumor size 600 mm³) in nude mice injected with humanglioblastoma cells U87 and treated with monoclonal antibody (MAb) 201F9,116F9 or phosphate buffered saline (PBS) (control).

FIG. 4 presents the Kaplan Meier plots of the growth of individualtumors (days until tumor size 600 mm³) in nude mice injected with humanglioblastoma cells U87 and treated with ionizing radiation (IR),monoclonal antibody 201F9 (MAb), simultaneously with ionizing radiationand monoclonal antibody 201F9 (IR+MAb) or phosphate buffered saline(PBS) (control). Ten mice in each group.

FIG. 5 shows a schematic presentation of localization of peptidesequences comprising the epitope for Mab 201F9 (SEQ ID NOs: 2-7) in the3D structure of YKL-40.

DETAILED DESCRIPTION OF THE INVENTION Antibody

It is an objective of the present invention to provide an antibody,antigen binding fragment or recombinant protein thereof capable ofrecognizing and selectively binding to an epitope on human YKL-40comprising at least one of the sequences selected from SEQ ID NOs:2-7and thereby inhibiting biological activity of YKL-40 associated withcell growth, cell differentiation, cell survival, and extracellulartissue remodeling processes.

Antibody molecules belong to a family of plasma proteins calledimmunoglobulins, whose basic building block, the immunoglobulin fold ordomain, is used in various forms in many molecules of the immune systemand other biological recognition systems. A typical immunoglobulin hasfour polypeptide chains, containing an antigen binding region known as avariable region and a non-varying region known as the constant region.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end. The constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains (Novotny J, & Haber E. ProcNatl Acad Sci USA. 82(14):4592-6, 1985).

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five (5) major classes of immunoglobulins: IgA, IgD,IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 andIgA-2. The heavy chains constant domains that correspond to thedifferent classes of immunoglobulins are called alpha (α), delta (δ),epsilon (ε), gamma (γ) and mu (μ), respectively. The light chains ofantibodies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino sequences of their constantdomain. The subunit structures and three-dimensional configurations ofdifferent classes of immunoglobulins are well known.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies. The variable domains are forbinding and determine the specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) also known as hypervariable regions both in the lightchain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four FR regions, largely a adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies. The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

An antibody that is contemplated for use in the present invention thuscan be in any of a variety of forms, including a whole immunoglobulin,an antibody fragment such as Fv, Fab, and similar fragments, a singlechain antibody which includes the variable domain complementaritydetermining regions (CDR), and the like forms, all of which fall underthe broad term “antibody”, as used herein. The present inventioncontemplates the use of any specificity of an antibody, polyclonal ormonoclonal, and is not limited to antibodies that recognize andimmunoreact with a specific antigen. In preferred embodiments, in thecontext of both the therapeutic and screening methods described below,an antibody or fragment thereof is used that is immunospecific for anantigen or epitope of the invention.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding fragments that are capable of cross-linkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

The term “antibody fragment” is used herein interchangeably with theterm “antigen binding fragment”.

Antibody fragments may be as small as about 4 amino acids, 5 aminoacids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 aminoacids, about 15 amino acids, about 17 amino acids, about 18 amino acids,about 20 amino acids, about 25 amino acids, about 30 amino acids ormore. In general, an antibody fragment of the invention can have anyupper size limit so long as it is has similar or immunologicalproperties relative to antibody that binds with specificity to anepitope comprising of amino acid residues 83-90, 96-105, 137-150,210-220, 304-314 and/or 318-329 of the SEQ ID NO: 1, or a peptidesequence selected from any of the sequences identified herein as SEQ IDNOs: 2-7, or a fragment of said sequences. Thus, in context of thepresent invention the term “antibodv fragment” is identical to term“antigen binding fragment”.

Antibody fragments retain some ability to selectively bind with itsantigen or receptor. Some types of antibody fragments are defined asfollows:

-   -   (1) Fab is the fragment that contains a monovalent        antigen-binding fragment of an antibody molecule. A Fab fragment        can be produced by digestion of whole antibody with the enzyme        papain to yield an intact light chain and a portion of one heavy        chain.    -   (2) Fab′ is the fragment of an antibody molecule can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain.        Two Fab′ fragments are obtained per antibody molecule.

Fab′ fragments differ from Fab fragments by the addition of a fewresidues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region.

-   -   (3) (Fab′)₂ is the fragment of an antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction.    -   (4) F(ab′)₂ is a dimer of two Fab′ fragments held together by        two disulfide bonds.

Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in a tight, non-covalentassociation (V_(H)-V_(L) dimer). It is in this configuration that thethree CDRs of each variable domain interact to define an antigen bindingsite on the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

-   -   (5) Single chain antibody (“SCA”), defined as a genetically        engineered molecule containing the variable region of the light        chain, the variable region of the heavy chain, linked by a        suitable polypeptide linker as a genetically fused single chain        molecule. Such single chain antibodies are also referred to as        “single-chain Fv” or “sFv” antibody fragments. Generally, the Fv        polypeptide further comprises a polypeptide linker between the        VH and VL domains that enables the sFv to form the desired        structure for antigen binding. For a review of sFv see Pluckthun        in The Pharmacology of Monoclonal Antibodies 113: 269-315        Rosenburg and Moore eds. Springer-Verlag, NY, 1994.

The term “diabodies” refers to a small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993).

The invention contemplate both polyclonal and monoclonal antibodyagainst human YKL-40, antigen binding fragments and recombinant proteinsthereof which possess at least one functional activity according to theinvention.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al. 1992. Production ofPolyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), pages1-5 (Humana Press); Coligan, et al., Production of Polyclonal Antiserain Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology,section 2.4.1, which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See,for exampie, Kohler & Milstein, Nature, 256:495-7 (1975); Coligan, etal., sections 2.5.1-2.6.7; and Harlow, et al., in: Antibodies: ALaboratory Manual, page 726, Cold Spring Harbor Pub. (1988), Monoclonalantibodies can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, e.g., Coligan, etal., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al.,Purification of Immunoglobulin G (IgG). In: Methods in MolecularBiology, 1992, 10:79-104, Humana Press, NY.

Methods of in vitro and in vivo manipulation of monoclonal antibodiesare well known to those skilled in the art. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,1975, Nature 256, 495-7, or may be made by recombinant methods, e.g., asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies for usewith the present invention may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., 1991,Nature 352: 624-628, as well as in Marks et al., 1991, J Mol Biol 222:581-597. Another method involves humanizing a monoclonal antibody byrecombinant means to generate antibodies containing human specific andrecognizable sequences. See, for review, Holmes, et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional polyclonal antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In additional to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567);Morrison et al., 1984, Proc Natl Acad Sci 81: 6851-6855.

Methods of making antibody fragments are also known in the art (see forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, NY, 1988, incorporated herein by reference). Antibodyfragments of the present invention can be prepared by proteolytichydrolysis of the antibody or by expression in E. coli of DNA encodingthe fragment. Antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies conventional methods. For example,antibody fragments can be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment canbe further cleaved using a thiol reducing agent, and optionally ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages, to produce 3.5S Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. These methods aredescribed, for example, in U.S. Pat. No. 4,036,945 and U.S. Pat. No.4,331,647, and references contained therein. These patents are herebyincorporated in their entireties by reference.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody. For example, Fv fragments comprise anassociation of V_(H) and V_(L) chains. This association may benoncovalent or the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde.Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains connected by an oligonucleotide.The structural gene is inserted into an expression vector, which issubsequently introduced into a host cell such as E. coli. Therecombinant host cells synthesize a single polypeptide chain with alinker peptide bridging the two V domains. Methods for producing sFvsare described, for example, by Whitlow, et al., 1991, In: Methods: ACompanion to Methods in Enzymology, 2:97; Bird et al., 1988, Science242:423-426; U.S. Pat. No. 4,946,778; and Pack, et al., 1993,BioTechnology 11:1271-77.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) are often involved in antigen recognition andbinding. CDR peptides can be obtained by cloning or constructing genesencoding the CDR of an antibody of interest. Such genes are prepared,for example, by using the polymerase chain reaction to synthesize thevariable region from RNA of antibody-producing cells. See, for example,Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2,page 106 (1991).

The invention contemplates human and humanized forms of non-human (e.g.murine) antibodies. Such humanized antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)that contain a minimal sequence derived from non-human immunoglobulin,such as the epitope recognising sequence. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a nonhuman species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. Humanized antibody(es) containing a minimalsequence(s) of antibody(es) of the invention, such as a sequence(s)recognising the epitope(s) described herein, is a preferred embodimentof the invention. In particular, the invention relates to humanizedforms of mouse anti-human YKL-40 monoclonal antibodies 201F9 and 116F9.

In some instances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are found neither in the recipientantibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, humanized antibodies will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., 1986, Nature321, 522-525; Reichmann et al., 1988, Nature 332, 323-329; Presta, 1992,Curr Op Struct Biol 2:593-596; Holmes et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The generation of antibodies may be achieved by any standard methods inthe art for producing polyclonal and monoclonal antibodies using anatural or recombinant human YKL-40 polypeptide or fragments thereof asan antigen. Such antibodies would be in a preferred embodiment generatedusing a naturally occurring or recombinantly produced human YKL-40(Swissprot Ass. No.: P36222) identified herein as SEQ ID NO: 1, orvariants or fragments thereof, or, in a more preferred embodiment, usingfragments of said polypeptides, said fragments being immunogenicfragments which meet at least two of the following criteria:

-   (i) being a contiguous amino acid sequence of at least 5 amino    acids;-   (ii) comprising an amino acid sequence derived from the sequence    identified as SEQ ID NO: 1 or-   (iii) comprising an amino acid sequence identified in SEQ ID NOs:    2-7, or a fragment thereof.

The antibodies may also be produced in vivo by the individual to betreated, for example, by administering an immunogenic fragment accordingto the invention to said individual. Accordingly, the present inventionfurther relates to a vaccine comprising an immunogenic fragmentdescribed above.

The application also relates to a method for producing an antibody ofthe invention said method comprising a step of providing of YKL-40 or animmunogenic fragment of YKL-40 described herein.

The invention relates to an antibody, which is capable of inhibitingbiological function of YKL-40 in connection with cell growth,differentiation, survival, metastatic potential and tissue remodeling,such as e.g. extracellular matrix remodelling with development offibrosis. In one embodiment such antibody may be an anti-human YKL-40antibody, or antibody fragment, or recombinant protein thereof, which iscapable of binding to an epitope comprising amino acid residues 83-90,96-105, 137-150, 210-220, 304-314 and/or 318-329 of the SEQ ID NO: 1, ora sequence selected from SEQ ID NOs: 2-7, or a fragment of saidsequences. Preferably, the epitope is located within the sequence YKL-40(SEQ ID NO: 1)

In another embodiment the invention relates to an antibody, which is notan anti-human YKL-40 antibody, said antibody being capable ofrecognising and binding to the epitope, which is recognised byanti-human YKL-40 antibody of above.

In still another embodiment the invention relates to a non-antibodycompound, which is capable of binding to the epitope described hereinand thereby inhibiting the function of YKL-40 protein in associationwith

-   -   development/progression of cancer growth, and/or    -   development/progression of cancer differentiation, and/or    -   development/progression of cancer survival, and/or    -   development/progression of cancer metastasis, and/or    -   development/progression of angiogenesis, and/or    -   development/progression of extracellular matrix remodeling,        and/or    -   development/progression of liver fibrosis, and/or    -   development/progression of lung fibrosis, and/or    -   development/progression of tissue fibrosis, and/or    -   development/progression of organ fibrosis, and/or    -   development of rheumatoid arthritis.

By the term “epitope” is meant the specific group of atoms (on anantigen molecule) that is recognized by (that antigen's) antibodies(thereby causing an immune response). The term “epitope” is theequivalent to the term “antigenic determinant”. The epitope may comprise3 or more amino acid residues, such as for example 4, 5, 6, 7, 8 aminoacid residues, located in close proximity, such as within a contiguousamino acid sequence, or located in distant parts of the amino acidsequence of an antigen, but due to protein folding have been approachedto each other.

The invention concerns an epitope which comprises at least 3 amino acidresidues selected

1) from amino acid residues 83-90, 96-105, 137-150, 210-220, 304-314and/or 318-329 of the SEQ ID NO: 1;2) consisting of a sequence selected from SEQ ID NOs: 2-7, or a fragmentthereof, or3) comprising a combination of amino acid residues derived from any ofthe sequences of SEQ ID NOs: 2-7.

In a preferred embodiment the epitope is located in human YKL-40, is of3 to 6 amino acid residues and comprises amino acid residues G, A, W, S,R, T and/or K.

Preferably, the invention concerns a monoclonal antibody against humanYKL-40 characterized by a capability of recognising of and binding tothe above epitope.

More particularly, the invention preferably concerns the group offunctionally active mouse anti-human YKL-40 monoclonal antibodiesidentified herein as monoclonal antibodies 115F9, 116F9 and 201F9.

According to the invention antibodies 115F9, 116F9 and 201F9 are capableof binding to an epitope as defined above and thereby inhibiting thefunction of YKL-40 in connection with cell growth and survival byinhibiting at least one biological activity of YKL-40 protein describedherein.

Functionally Active Antibody

Thus, the present invention relates to an antibody, an antigen bindingfragment, or recombinant protein thereof, which is capable ofrecognising an epitope comprising or consisting of a sequence selectedfrom any of the sequences identified as SEQ ID NOs: 2-7, of fragmentsthereof, said antibody, antigen binding fragment or recombinant proteinbeing capable of modulating at least one biological activity of humanYKL-40 or an YKL-40 functional homolog, or a functional fragmentthereof, said activity being associated with i) cell growth ii) cellsurvival, iii) cell differentiation iv) extracellular matrix remodeling,v) development of liver fibrosis, vi) development of tissue fibrosis,vii) development of organ fibrosis, viii) development of angiogenesis,ix) development of rheumatoid arthritis, x) development of inflammation,and/or xi) development of metastasis.

In a preferred embodiment, the antibody is an anti-human YKL-40monoclonal antibody.

The term “cell growth” is interchangeably used herein with the term“cell proliferation” and designates the phenomenon of great increase incell number due to cell division.

The term “cell survival” is designate the phenomenon of cell continuingto live or exist, especially after coming close to dying or beingdestroyed, or after being improperly treated, for example as withfactors affecting cell homeostasis so that inducing cell selfdestruction due to apoptosis. The wording “inhibition/stimulation cellsurvival” is interchangeably used herein with the wording“stimulation/inhibition of apoptosis/cell death”

In the present content by the wording “YKL-40 functional homolog” ismeant a polypeptide

-   -   comprising one of the immunogenic fragments of human YKL-40        defined above,    -   capable of at least one of YKL-40 biological activities        associated with cell growth, survival, differentiation,        apoptosis, angiogenesis, extracellular matrix remodeling,        development of metastasis, development of liver fibrosis,        development of tissue fibrosis, development of organ fibrosis,        development of rheumatoid arthritis and/or development of        inflammation    -   being recognizable by an antibody of the invention, preferably        monoclonal antibody 115F9, 116F9 and/or 201F9.

In the present content by the term “modulating” is meant that anantibody, an antigen binding fragment, or recombinant protein thereof,is capable of enhancing or diminishing biological activity of humanYKL-40, or a functional homolog thereof, or a biologically activefragment thereof. In preferred embodiment the invention features anantibody, antigen binding fragment, or recombinant protein thereof,which is capable of modulating of at least one biological activity ofYKL-40, such as stimulating of cell proliferation, cell growth, celldifferentiation, cell survival, modulation of adhesion and/or motilityof cells. In a preferred embodiment the cells are tumor cells. The tumorcells are in preferred embodiment cancer cells or hematologicalmalignant cells. The cancer cells may be from either primary ormetastatic cancer.

By “primary cancer” is meant a group of tumor cells, which have acquiredat least one characteristic feature of cancer cells, however have notyet invaded the neighbouring tissues and hold together in a tumorlocalized at the place of primary origin. By “metastatic cancer” ismeant a group of tumor cells, which originate from the cells of aprimary cancer, which have invaded the tissue surrounding said primarycancer, disseminated through the body, adhered at a new distant placeand grown to a new tumor.

The examples of primary and metastatic cancers of the present inventioninclude, but is not limited by carcinoma of the breast, colorectal,pancreas, stomach, GIST, hepatocellular, lung, small cell lung, ovarian,uterine, cervix, bladder, renal, prostate, testis, thyroid carcinoma,malignant melanoma, osteosarcoma, chondrosarcoma, myosarcoma,glioblastoma or other brain tumors, head/neck other gastrointestinal andgerm cell tumors, and haematologic malignancies.

A functional antibody according to invention is capable of modulatingproliferation, differentiation, and/or influencing cell survival of bothprimary and metastatic cancer cells, preferably inhibiting cellgrowth/proliferation/differentiation of said cells and/orinducing/stimulating apotosis/cell death of said cells. In otherembodiments, functional antibodies of the invention are also capable ofmodulating proliferation, and/or influencing cell survival of non-cancercells such as endothelial, fibroblast, synovial, chondrocyte, bloodcells (e.g. monocytes/macrophages, neutrophils and precurcors), hepaticstellate cells, vascular smooth muscle, epithelial cells, as abiological activity of human YKL-40 has previously been shown play animportant role in stimulation of proliferation and survival of thesecells. The example of such YKL-40 biological activity may be selectedfrom, but not limited by capability of YKL-40 of

i) binding of chitin,ii) binding of heparin,iii) binding of heparan sulphate,iv) binding of hyaluronan,v) binding of long and/or short oligosaccharides,vi) serving as a cellular growth factor,vii) serving as a chemo-attractant for cells,viii) interfering with the synthesis of hyaluronan,ix) stimulating signal transduction pathways associated with cellsurvival,x) stimulating differentiation,xi) stimulating angiogenesis,xii) stimulating fibrogenesis,xiii) stimulating extracellular matrix remodelling and/orxiv) stimulating inflammation.

Thus, a functionally active antibody of the invention, an antigenbinding fragment-thereof, or recombinant protein thereof, whichspecifically recognises an epitope(s) described herein, is capable ofmodulating any of the above biological activity of YKL-40, preferably,any activity, which is associated with the processes influencing cellproliferation or survival.

Antibodies 115F9, 116F9 and 201F9 are the preferred functionally activeantibodies of the invention capable of modulating any one of the YKL-40activities described above, or two or more of the activities.

Peptide Fragments

An epitope of the invention may be represented by 3 to 6 amino acidresidues of the SEQ ID NO: 1, said amino acid residues are preferablylocated in the areas of YKL-40 comprising amino acid residues 83-90,96-105, 137-150, 210-220, 304-314 and/or 318-329 according to thesequence of SEQ ID NO: 1, wherein the preferred amino acid residuesbeing G, A, W, S, R, T and/or K. The epitope may also be represented bya contiguous amino acid sequence derived from the sequence of SEQ ID NO:1, such as a sequence selected from SEQ ID NOs: 2-7 or a fragmentthereof.

Thus, one aspect of the invention concerns the epitope(s) comprising orconsisting of at least one of the sequences identified below:

LKNRNPNL (SEQ ID NO: 2) VGGWN FGSQR (SEQ ID NO: 3) LAWLYPGRRDKQHF (SEQID NO: 4) GAWRGTTGHHS (SEQ ID NO: 5) RGATVHRTLGQ. (SEQ ID NO: 6)YATKGNQWVGY, (SEQ ID NO: 7)ora fragment thereof, or a variant thereof.

The above sequences according to the invention are immunogenic fragmentsof human YKL-40, and an epitope comprising or consisting of at least oneof these sequences is recognized by a functional antibody of theinvention of above. Thus, in one aspect the peptide fragments identifiedabove may be used for raising a functional antibody of the invention.

In another aspect of the invention, the peptide fragments may be used asalternative ligands of the YKL-40 receptor capable of competing for thebinding sites on the receptor with YKL-40 protein. Competitive bindingof the peptide fragments to the YKL-40 receptor may attenuate thefunction of YKL-40 executed through the receptor binding and activation.Thus, the peptide fragments may be used as compounds for the inhibitingthe functions of YKL-40 protein executed through the receptor binding.Preferably, the peptide fragments are to be used for inhibiting thefunction of YKL-40 associated with the processes of

-   -   i) stimulating of cell growth,    -   ii) stimulating of cell survival,    -   iii) stimulation of cell proliferation,    -   iv) stimulating of cell differentiation,    -   v) stimulating of extracellular matrix remodeling,    -   vi) stimulating of development of liver fibrosis,    -   vii) stimulating of development of organ fibrosis,    -   viii) stimulation of development of tissue fibrosis,    -   ix) stimulating of development of rheumatoid arthritis,    -   x) stimulation of development of metastasis,    -   xi) stimulating of angiogenesis, and/or    -   xii) stimulation of inflammation.

In the present application the standard one-letter code for amino acidresidues, as well as the standard three-letter code are applied.Abbreviations for amino acids are in accordance with the recommendationsin the IUPAC-IUB Joint Commission on Biochemical Nomenclature Eur. J.Biochem, 1984, vol. 184, pp 9-37. Throughout the description and claimseither the three letter code or the one letter code for natural aminoacids are used. Where the L or D form has not been specified it is to beunderstood that the amino acid in question has the natural L form, cf.Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984) or the D form, so thatthe peptides formed may be constituted of amino acids of L form, D form,or a sequence of mixed L forms and D forms.

Where nothing is specified it is to be understood that the C-terminalamino acid of a peptide of the invention exists as the free carboxylicacid, this may also be specified as “—OH”. However, the C-terminal aminoacid of a compound of the invention may be the amidated derivative,which is indicated as “—NH₂”. Where nothing else is stated theN-terminal amino acid of a polypeptide comprise a free amino-group, thismay also be specified as “H—”.

Where nothing else is specified amino acid can be selected from anyamino acid, whether naturally occurring or not, such as alfa aminoacids, beta amino acids, and/or gamma amino acids. Accordingly, thegroup comprises but are not limited to: Ala, Val, Leu, Ile, Pro, Phe,Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, HisAib, Nal, Sar, Orn, Lysine analogues, DAP, DAPA and 4Hyp.

Basic amino acid residues are according to invention represented by theresidues of amino acids Arg, Lys, and His, acidic amino acid residues—bythe residues of amino acids Glu and Asp. Basic and amino acid residuesconstitute a group of charged amino acid residues. The group ofhydrophobic amino acid residues is represented by the residues of aminoacids Leu, Ile, Val, Phe, Trp, Tyr, and Met.

According to the invention modifications of the peptides may beperformed, such as for example glycosylation and/or acetylation of theamino acids.

In one embodiment variants may be understood as exhibiting amino acidsequences gradually differing from the preferred predetermined sequence,as the number and scope of insertions, deletions and substitutionsincluding conservative substitutions increase. This difference ismeasured as a reduction in homology between the predetermined sequenceand the variant.

According to the invention a peptide sequence may have the length of 5or more amino acid residues. The length of 6-8 amino acid residues ispreferred, as the epitope/immunogenic fragment of the inventionpreferably comprises 6-8 amino acid residues. However, immunogenicfragments of the above sequences consisting of 3 to 7 amino acidresidues such as for example 4, 5, or 6, are also within the scope ofthe invention.

The upper limit for the number of amino acid residues in a peptidefragment of the invention may vary. Thus, a peptide fragment comprisingan immunogenic fragment of the invention may have the length up to 250amino acid residues. For example it may comprise from 5 to 150 aminoacid residues, such as 5 to 125, for example 5 to 100, such as 5 to 80,for example 5 to 65, 5 to 50, 5 to 30 or 5 to 20.

Thus, the invention also features peptide fragments comprising orconsisting of 8 to 25 amino acid residues, such as 8 to 20, for example8 to 15. In other embodiments the length of a peptide fragment may befrom 10 to 25, such as 12 to 25, for example from 14 to 25, or it may befrom 14 to 20 or from 14 to 18 amino acid residues. In some embodiments,a peptide fragment comprising the immunogenic sequence of the invention(any of SEQ ID NO: 2-7) may consists of more then 25 amino acidresidues, such as from 26 to 50 amino acid residues, for example 28-30,31-35, 36-40, 41-45 or 46-49 amino acid residues.

It is understood that all the above peptide fragments comprise orconsists of a least one of the sequences selected from SEQ ID NO: 2-7,or a fragment or variant thereof.

A peptide fragment of the invention may comprise or consist of more thanone immunogenic sequence of SEQ ID NOs: 2-7 or fragments or variantsthereof. The peptide fragments may be formulated as monomers. This meansthat they may be represented by single copies of individual peptidesequences comprising the immunogenic sequence. A peptide fragment mayalso comprise or consist of more than one copy of the same sequence.Thus, the invention also relates to polymers of individual peptidesequences of the above. A polymer of a peptide sequence may berepresented by a single peptide chain, wherein an individual peptidesequence is repeated two or more times in the chain, or it may be amolecule, wherein copies of individual peptide sequence are connected toeach other via a linker group. Non-limited examples of such polymers maybe dendrimeric polymers, wherein individual copies of a peptide sequenceare attached to a core molecule, such as lysine.

A compound may comprise or consist of two or more different immunogenicsequences of the invention.

The peptide fragments comprising immunogenic sequences of the inventionmay comprise or consist of variants of said sequences.

“Variants of peptide sequences” means that the peptides may be modified,for example by substitution of one or more of the amino acid residues.Both L-amino acids and D-amino acids may be used. Other modification maycomprise derivatives such as esters, sugars, etc. Examples are methyland acetyl esters. Polymerisation such as repetitive sequences orattachment to various carriers are well-known in the art, e.g. lysinebackbones, such as lysine dendrimers carrying 4 peptides, 8 peptides, 16peptides, or 32 peptides. Other carriers may be protein moieties, suchas bovine serum albumin (BSA), or lipophilic dendrimers, or micelle-likecarriers formed by lipophilic derivatives, or starburst (star-like)carbon chain polymer conjugates, or ligand presenting assembly (LPA)based on derivatives of diethylaminomethane.

Variants of the peptide fragments according to the invention maycomprise, within the same variant, or fragments thereof or amongdifferent variants, or fragments thereof, at least one substitution,such as a plurality of substitutions introduced independently of oneanother. Variants of the complex, or fragments thereof may thus compriseconservative substitutions independently of one another, wherein atleast one glycine (Gly) of said variant, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Ala, Val, Leu, and Ile, and independently thereof,variants, or fragments thereof, wherein at least one alanine (Ala) ofsaid variants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Gly, Val, Leu, andIle, and independently thereof, variants, or fragments thereof, whereinat least one valine (Val) of said variant, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Gly, Ala, Leu, and Ile, and independently thereof,variants, or fragments thereof, wherein at least one leucine (Leu) ofsaid variant, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Gly, Ala, Val, andIle, and independently thereof, variants, or fragments thereof, whereinat least one isoleucine (Ile) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Gly, Ala, Val and Leu, and independently thereof,variants, or fragments thereof wherein at least one aspartic acids (Asp)of said variant, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Glu, Asn, and Gln,and independently thereof, variants, or fragments thereof, wherein atleast one aspargine (Asn) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Asp, Glu, and Gln, and independently thereof, variants, orfragments thereof, wherein at least one glutamine (Gln) of saidvariants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Asp, Glu, and Asn,and wherein at least one phenylalanine (Phe) of said variants, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Tyr, Trp, His, Pro, and preferablyselected from the group of amino acids consisting of Tyr and Trp, andindependently thereof, variants, or fragments thereof, wherein at leastone tyrosine (Tyr) of said variants, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofPhe, Trp, His, Pro, preferably an amino acid selected from the group ofamino acids consisting of Phe and Trp, and independently thereof,variants, or fragments thereof, wherein at least one arginine (Arg) ofsaid fragment is substituted with an amino acid selected from the groupof amino acids consisting of Lys and His, and independently thereof,variants, or fragments thereof, wherein at least one lysine (Lys) ofsaid variants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Arg and His, andindependently thereof, variants, or fragments thereof, and independentlythereof, variants, or fragments thereof, and wherein at least oneproline (Pro) of said variants, or fragments thereof is substituted withan amino acid selected from the group of amino acids consisting of Phe,Tyr, Trp, and His, and independently thereof, variants, or fragmentsthereof, wherein at least one cysteine (Cys) of said variants, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Asp, Glu, Lys, Arg, His, Asn, Gln,Ser, Thr, and Tyr.

It thus follows from the above that the same functional equivalent of apeptide fragment, or fragment of said functional equivalent may comprisemore than one conservative amino acid substitution from more than onegroup of conservative amino acids as defined herein above. The term“conservative amino acid substitution” is used synonymously herein withthe term “homologous amino acid substitution”. The groups ofconservative amino acids are as the following:

P, A, G, S, T (neutral, weakly hydrophobic)Q, N, E, D, B, Z (hydrophilic, acid amine)H, K, R (hydrophilic, basic)L, I, V, M, F, Y, W (hydrophobic, aromatic)C (cross-link forming)

Conservative substitutions may be introduced in any position of apreferred predetermined peptide of the invention or fragment thereof. Itmay however also be desirable to introduce non-conservativesubstitutions, particularly, but not limited to, a non-conservativesubstitution in any one or more positions.

A non-conservative substitution leading to the formation of afunctionally equivalent fragment of the peptide of the invention wouldfor example differ substantially in polarity, for example a residue witha non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met)substituted for a residue with a polar side chain such as Gly, Ser, Thr,Cys, Tyr, Asn, or GIn or a charged amino acid such as Asp, Glu, Arg, orLys, or substituting a charged or a polar residue for a non-polar one;and/or ii) differ substantially in its effect on peptide backboneorientation such as substitution of or for Pro or Gly by anotherresidue; and/or iii) differ substantially in electric charge, forexample substitution of a negatively charged residue such as Glu or Aspfor a positively charged residue such as Lys, His or Arg (and viceversa); and/or iv) differ substantially in steric bulk, for examplesubstitution of a bulky residue such as His, Trp, Phe or Tyr for onehaving a minor side chain, e.g. Ala, Gly or Ser (and vice versa).

Substitution of amino acids may in one embodiment be made based upontheir hydrophobicity and hydrophilicity values and the relativesimilarity of the amino acid side-chain substituents, including charge,size, and the like. Exemplary amino acid substitutions, which takevarious of the foregoing characteristics into consideration, are wellknown to those of skill in the art and include: arginine and lysine;glutamate and aspartate; serine and threonine; glutamine and asparagine;and valine, leucine and isoleucine.

The function of YKL-40 which is associated with the processes of

-   -   i) stimulation of cell growth, and/or    -   ii) stimulation of cell proliferation, and/or    -   iii) stimulation of cell survival, and/or    -   iv) stimulation of cell differentiation, and/or    -   v) stimulation of development of metastases, and/or    -   vi) stimulation of extracellular matrix remodeling, and/or    -   vii) stimulation of development of liver fibrosis, and/or    -   viii) stimulation of development of organ fibrosis, and/or    -   ix) stimulation of development of tissue fibrosis and/or    -   x) stimulation of development of rheumatoid arthritis, and/or    -   xi) stimulation of angiogenesis, and/or    -   xii) stimulation of inflammation        may by selected from the following biological activities of        YKL-40    -   i) binding of chitin, and/or    -   ii) binding of heparin, and/or    -   iii) binding of heparin sulphate and/or    -   iv) binding of hyaluronan, and/or    -   v) binding of long and/or short oligosaccharides, and/or    -   vi) serving as a cellular growth factor, and/or    -   vii) serving as a cellular differentiation factor, and/or    -   viii) serving as a chemo-attractant for cells, and/or    -   ix) interfering with the synthesis of hyaluronan, and/or    -   x) interfering with extracellular matrix remodeling and/or    -   xi) stimulating signal transduction pathways associated with        cell survival, and/or    -   xii) stimulating angiogenesis, and/or    -   xiii) interfering with inflammation, and/or    -   xiv) stimulating fibrogenesis.

These biological functions of YKL-40 are according to the inventionexecuted involving the structural areas of the protein, which compriseor consist of at least one of the following sequences

LKNRNPNL (SEQ ID NO: 2) VGGWN FGSQR (SEQ ID NO: 3) LAWLYPGRRDKQHF (SEQID NO: 4) GAWRGTTGHHS (SEQ ID NO: 5) RGATVHRTLGQ. (SEQ ID NO: 6)YATKGNQWVGY (SEQ ID NO: 7)

According to the invention, the SEQ ID NOs: 2-7 all or at least one ofthem are involved in formation of a receptor binding site on YKL-40,said site being involved in execution at least one of the aboveidentified biological activities of YKL-40. Therefore, the inventionconsiders the presence any of the sequences of SEQ ID NOs: 2-7 in alonger peptide sequence to be essential for said longer peptide sequencepossesses the capability to competitive (i) binding to the YKL-40receptor, and/or (ii) binding to the functionally active antibody of theinvention. Thus, a sequence which includes at least one of the sequencesof SEQ ID NOs: 2-7 is concerned by the invention as a modulator ofbiological activity of human YKL-40 in vivo and in vitro. Thus, whensuch sequence competes with YKL-40 for the receptor binding, thesequence may thereby attenuate the activity of YKL-40, such asdown-regulate stimulation of cell proliferation, differentiation and/orcell survival induced by YKL-40, or when compete for the antibodybinding, the sequence may attenuate the inhibiting activity of theantibody and thereby up-regulate stimulation of cell proliferationand/or cell survival dependent on YKL-40. Accordingly, a peptidesequence of 8-200 amino acids long comprising a sequence selected forSEQ ID NOs: 2-7, or a fragment thereof, or a variant thereof, isconsidered to be a functional homologue of human YKL-40, or functionalhomologue any of the sequences identified as SEQ ID NOs: 2-7.

Production of Individual Peptide Sequences

The peptide sequences of the present invention may be prepared by anyconventional synthetic methods, recombinant DNA technologies, enzymaticcleavage of full-length proteins which the peptide sequences are derivedfrom, or a combination of said methods.

Recombinant Preparation

Thus, in one embodiment the peptides of the invention are produced byuse of recombinant DNA technologies.

The DNA sequence encoding a peptide or the corresponding full-lengthprotein the peptide originates from may be prepared synthetically byestablished standard methods, e.g. the phosphoamidine method describedby Beaucage and Caruthers, 1981, Tetrahedron Lett. 22:1859-1869, or themethod described by Matthes et al., 1984, EMBO J. 3:801-805. Accordingto the phosphoamidine method, oligonucleotides are synthesised, e.g. inan automatic DNA synthesiser, purified, annealed, ligated and cloned insuitable vectors.

The DNA sequence encoding a peptide may also be prepared byfragmentation of the DNA sequences encoding the correspondingfull-length protein of peptide origin, using DNAase I according to astandard protocol (Sambrook et al., Molecular cloning: A Laboratorymanual. 2 rd ed., CSHL Press, Cold Spring Harbor, N.Y., 1989). Thepresent invention relates to full-length proteins selected from thegroups of proteins identified above. The DNA encoding the full-lengthproteins of the invention may alternatively be fragmented using specificrestriction endonucleases. The fragments of DNA are further purifiedusing standard procedures described in Sambrook et al., Molecularcloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor,N.Y., 1989.

The DNA sequence encoding a full-length protein may also be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of thefull-length protein by hybridisation using synthetic oligonucleotideprobes in accordance with standard techniques (cf. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989). The DNA sequence may also be prepared by polymerase chainreaction using specific primers, for instance as described in U.S. Pat.No. 4,683,202 or Saiki et al., 1988, Science 239:487-491.

The DNA sequence is then inserted into a recombinant expression vector,which may be any vector, which may conveniently be subjected torecombinant DNA procedures. The choice of vector will often depend onthe host cell into which it is to be introduced. Thus, the vector may bean autonomously replicating vector, i.e. a vector that exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

In the vector, the DNA sequence encoding a peptide or a full-lengthprotein should be operably connected to a suitable promoter sequence.The promoter may be any DNA sequence, which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.Examples of suitable promoters for directing the transcription of thecoding DNA sequence in mammalian cells are the SV 40 promoter (Subramaniet al., 1981, Mol. Cell. Biol. 1:854-864), the MT-1 (metallothioneingene) promoter (Palmiter et al., 1983, Science 222: 809-814) or theadenovirus 2 major late promoter. A suitable promoter for use in insectcells is the polyhedrin promoter (Vasuvedan et al., 1992, FEBS Lett.311:7-11). Suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol.Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. Mol. Appl. Gen. 1:419-434) or alcohol dehydrogenase genes (Young et al., 1982, in GeneticEngineering of Microorganisms for Chemicals, Hollaender et al, eds.,Plenum Press, New York), or the TPI1 (U.S. Pat. No. 4,599,311) orADH2-4c (Russell et al., 1983, Nature 304:652-654) promoters. Suitablepromoters for use in filamentous fungus host cells are, for instance,the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or thetpiA promoter.

The coding DNA sequence may also be operably connected to a suitableterminator, such as the human growth hormone terminator (Palmiter etal., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op.cit.) or ADH3 (McKnight et al., op. cit.) promoters. The vector mayfurther comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 EIb region), transcriptional enhancer sequences(e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. theones encoding adenovirus VA RNAs).

The recombinant expression vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence (when the host cell is a mammalian cell) isthe SV 40 origin of replication. The vector may also comprise aselectable marker, e.g. a gene the product of which complements a defectin the host cell, such as the gene coding for dihydrofolate reductase(DHFR) or one which confers resistance to a drug, e.g. neomycin,hydromycin or methotrexate.

The procedures used to ligate the DNA sequences coding the peptides orfull-length proteins, the promoter and the terminator, respectively, andto insert them into suitable vectors containing the informationnecessary for replication, are well known to persons skilled in the art(cf., for instance, Sambrook et al., op.cit.).

To obtain recombinant peptides of the invention the coding DNA sequencesmay be usefully fused with a second peptide coding sequence and aprotease cleavage site coding sequence, giving a DNA construct encodingthe fusion protein, wherein the protease cleavage site coding sequencepositioned between the HBP fragment and second peptide coding DNA,inserted into a recombinant expression vector, and expressed inrecombinant host cells. In one embodiment, said second peptide selectedfrom, but not limited by the group comprising glutathion-5-reductase,calf thymosin, bacterial thioredoxin or human ubiquitin natural orsynthetic variants, or peptides thereof. In another embodiment, apeptide sequence comprising a protease cleavage site may be the FactorXa, with the amino acid sequence IEGR, enterokinase, with the amino acidsequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, orAcharombacter lyticus, with the amino acid sequence XKX, cleavage site.

The host cell into which the expression vector is introduced may be anycell which is capable of expression of the peptides or full-lengthproteins, and is preferably a eukaryotic cell, such as invertebrate(insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes ormammalian cells, in particular insect and mammalian cells. Examples ofsuitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCCCRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL-61) celllines. Methods of transfecting mammalian cells and expressing DNAsequences introduced in the cells are described in e.g. Kaufman andSharp, J. Mol. Biol. 159, 1982, pp. 601-621; Southern and Berg, 1982, J.Mol. Appl. Genet. 1:327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci.USA 79: 422-426; Wigler et al., 1978, Cell 14:725; Corsaro and Pearson,1981, in Somatic Cell Genetics 7, p. 603; Graham and van der Eb, 1973,Virol. 52:456; and Neumann et al., 1982, EMBO J. 1:841-845.

Alternatively, fungal cells (including yeast cells) may be used as hostcells. Examples of suitable yeast cells include cells of Saccharomycesspp. or Schizosaccharomyces spp., in particular strains of Saccharomycescerevisiae. Examples of other fungal cells are cells of filamentousfungi, e.g. Aspergillus spp. or Neurospora spp., in particular strainsof Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp.for the expression of proteins is described in, e.g., EP 238 023.

The medium used to culture the cells may be any conventional mediumsuitable for growing mammalian cells, such as a serum-containing orserum-free medium containing appropriate supplements, or a suitablemedium for growing insect, yeast or fungal cells. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g. in catalogues of the American Type CultureCollection).

The peptides or full-length proteins recombinantly produced by the cellsmay then be recovered from the culture medium by conventional proceduresincluding separating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulphate,purification by a variety of chromatographic procedures, e.g. HPLC, ionexchange chromatography, affinity chromatography, or the like.

Synthetic Preparation

The methods for synthetic production of peptides are well known in theart. Detailed descriptions as well as practical advice for producingsynthetic peptides may be found in Synthetic Peptides: A User's Guide(Advances in Molecular Biology), Grant G. A. ed., Oxford UniversityPress, 2002, or in: Pharmaceutical Formulation: Development of Peptidesand Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.

Peptides may for example be synthesised by using Fmoc chemistry and withAcm-protected cysteins. After purification by reversed phase HPLC,peptides may be further processed to obtain for example cyclic or C- orN-terminal modified isoforms. The methods for cyclization and terminalmodification are well-known in the art and described in detail in theabove-cited manuals.

In a preferred embodiment the peptide sequences of the invention areproduced synthetically, in particular, by the Sequence Assisted PeptideSynthesis (SAPS) method.

By SAPS peptides may be synthesised either batchwise in a polyethylenevessel equipped with a polypropylene filter for filtration or in thecontinuous-flow version of the polyamide solid-phase method (Dryland, A.and Sheppard, R. C., (1986) J. Chem. Soc. Perkin Trans. I, 125-137.) ona fully automated peptide synthesiser using 9-fluorenylmethyloxycarbonyl(Fmoc) or tert.-Butyloxycarbonyl, (Boc) as N-a-amino protecting groupand suitable common protection groups for side-chain functionality. Whensynthesised, individual peptide sequences may then be formulated asmultimers using well-known in the art techniques, for examples dimers ofthe sequences may be obtained by the LPA method described in WO00/18791, denrimeric polymers by the MAP synthesis are described inPCT/US90/02039.

Pharmaceutical Composition

In the present context the term “pharmaceutical composition” is usedsynonymously with the term “medicament”.

In some embodiments the invention concerns a pharmaceutical compositioncomprising an antibody capable of binding the epitope as defined above,antigen binding fragment or recombinant protein thereof. Preferably, apharmaceutical composition comprises monoclonal antibodies 115F9, 116F9and/or 201F9, antigen binding fragment or recombinant protein thereof,most preferably monoclonal antibody 201F9, antigen binding fragment orrecombinant protein thereof. Most preferably, a pharmaceuticalcomposition of the invention comprises or essentially comprises ahumanized form(s) of antibodies 115F9, 116F9 and/or 201F9.

In other embodiments the invention concerns a pharmaceutical compositioncomprising an immunogenic fragment of YKL-40, said fragment comprisingat least one of the sequences identified as SEQ ID NOs: 2-7.

In one embodiment a pharmaceutical composition comprises the peptidesequence identified as SEQ ID NO: 2, or a fragment thereof, or a variantthereof. In another embodiment the composition comprises the peptidesequence identified as SEQ ID NO: 3, or a fragment thereof, or a variantthereof. In still another embodiment the composition comprises thepeptide sequence identified as SEQ ID NO: 4, or a fragment thereof, or avariant thereof. In yet another embodiment the composition comprises thepeptide sequence identified as SEQ ID NO: 5, or a fragment thereof, or avariant thereof. In still yet another embodiment the compositioncomprises the peptide sequence identified as SEQ ID NO: 6, or a fragmentthereof, or a variant thereof, or the peptide sequence identified as SEQID NO: 6, or a fragment thereof, or a variant thereof. In someembodiments a pharmaceutical composition may comprise any combination ofthe sequences SEQ ID NOs.: 2-7.

In a composition the peptide sequences may be formulated as isolatedindividual peptide fragments or multimers or dimers thereof as discussedabove.

The pharmaceutical composition described above may for example be usedto promote death of cancer cells in vitro or in vivo.

The composition is administered to a subject in vivo or to be used invitro contains an effective amount of one or more of the compoundsdescribed above in combination with pharmaceutically acceptableadditives. Such medicament may suitably be formulated for oral,percutaneous, intramuscular, intravenous, intracranial, intrathecal,intracerebroventricular, intranasal or pulmonal administration.

Strategies in formulation development of medicaments and compositionsbased on the compounds of the present invention generally correspond toformulation strategies for any other protein-based drug product.Potential problems and the guidance required to overcome these problemsare dealt with in several textbooks, e.g. “Therapeutic Peptides andProtein Formulation. Processing and Delivery Systems”, Ed. A. K. Banga,Technomic Publishing AG, Basel, 1995.

Injectables are usually prepared either as liquid solutions orsuspensions, solid forms suitable for solution in, or suspension in,liquid prior to injection. The preparation may also be emulsified. Theactive ingredient is often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol or the like, and combinations thereof. In addition, if desired,the preparation may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents, or which enhancethe effectiveness or transportation of the preparation.

Formulations of the compounds of the invention can be prepared bytechniques known to the person skilled in the art. The formulations maycontain pharmaceutically acceptable carriers and excipients includingmicrospheres, liposomes, microcapsules, nanoparticles or the like.

The preparation may suitably be administered by injection, optionally atthe site, where the active ingredient is to exert its effect. Additionalformulations which are suitable for other modes of administrationinclude suppositories, nasal, pulmonal and, in some cases, oralformulations. For suppositories, traditional binders and carriersinclude polyalkylene glycols or triglycerides. Such suppositories may beformed from mixtures containing the active ingredient(s) in the range offrom 0.5% to 10%, preferably 1-2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and generally contain 10-95% of theactive ingredient(s), preferably 25-70%.

Other formulations are such suitable for nasal and pulmonaladministration, e.g. inhalators and aerosols.

The active compound may be formulated as neutral or salt forms.Pharmaceutically acceptable salts include acid addition salts (formedwith the free amino groups of the peptide compound) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic acid, oxalic acid, tartaric acid,mandelic acid, and the like. Salts formed with the free carboxyl groupmay also be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The preparations are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective.The quantity to be administered depends on the subject to be treated,including, e.g. the weight and age of the subject, the disease to betreated and the stage of disease. Suitable dosage ranges are per kilobody weight normally of the order of several hundred μg activeingredient per administration with a preferred range of from about 0.1μg to 5000 μg per kilo body weight. Using monomeric forms of thecompounds, the suitable dosages are often in the range of from 0.1 μg to5000 μg per kilo body weight, such as in the range of from about 0.1 μgto 3000 μg per kilo body weight, and especially in the range of fromabout 0.1 μg to 1000 μg per kilo body weight. Using multimeric forms ofthe compounds, the suitable dosages are often in the range of from 0.1μg to 1000 μg per kilo body weight, such as in the range of from about0.1 μg to 750 μg per kilo body weight, and especially in the range offrom about 0.1 μg to 500 μg per kilo body weight such as in the range offrom about 0.1 μg to 250 μg per kilo body weight. In particular whenadministering nasally smaller dosages are used than when administeringby other routes. Administration may be performed once or may be followedby subsequent administrations. The dosage will also depend on the routeof administration and will vary with the age and weight of the subjectto be treated. A preferred dosage of multimeric forms would be in theinterval 1 mg to 70 mg per 70 kg body weight.

For some indications a localised or substantially localised applicationis preferred.

Some of the compounds of the present invention are sufficiently active,but for some of the others, the effect will be enhanced if thepreparation further comprises pharmaceutically acceptable additivesand/or carriers. Such additives and carriers will be known in the art.In some cases, it will be advantageous to include a compound, whichpromotes delivery of the active substance to its target.

In many instances, it will be necessary to administrate the formulationmultiple times. Administration may be a continuous infusion, such asintraventricular infusion or administration in more doses such as moretimes a day, daily, more times a week, weekly, etc. It is preferred thatadministration of the medicament is initiated before or shortly afterthe individual has been subjected to the factor(s) that may lead to celldeath. Preferably the medicament is administered within 8 hours from thefactor onset, such as within 5 hours from the factor onset. Many of thecompounds exhibit a long term effect whereby administration of thecompounds may be conducted with long intervals, such as 1 week or 2weeks.

In connection with the use in nerve guides, the administration may becontinuous or in small portions based upon controlled release of theactive compound(s). Furthermore, precursors may be used to control therate of release and/or site of release. Other kinds of implants and wellas oral administration may similarly be based upon controlled releaseand/or the use of precursors.

The present invention also relates to treatment of individuals in needfor inducing cell death, inhibiting cell growth development of liverfibrosis or rheumatoid arthritis. The treatment involves administeringan pharmaceutical composition of the invention comprising an effectiveamount of one or more compounds as defined above.

Treatment

As discussed above, antibodies and peptide fragments described hereinmay be used for modulating, such as inhibiting or stimulating, a leastone of YKL-40 biological activities listed below:

-   -   i) binding of chitin,    -   ii) binding of heparin,    -   iii) binding of heparan sulphate,    -   iv) binding of hyaluronan,    -   v) binding of long and/or short oligosaccharides,    -   vi) serving as a cellular growth factor,    -   vii) serving as a differentiation factor,    -   viii) serving as a chemo-attractant for cells,    -   ix) interfering with the synthesis of hyaluronan,    -   x) stimulating signal transduction pathways associated with cell        survival,    -   xi) stimulating angiogenesis,    -   xii) stimulating fibrogenesis,    -   xiii) interfering with extracellular matrix remodeling,    -   xiv) interfering with inflammation, and/or    -   xv) stimulating development of metastasis

The above activities of human YKL-40 have been associated with thecapability of the protein to stimulate cell growth, proliferation,differentiation and survival, extracellular matrix remodeling, fibrosisand angiogenesis. Therefore, antibodies and peptide fragments of theinvention may also be used for inhibiting cell growth, proliferation,differentiation and survival, development of metastases and/orextracellular matrix remodeling, fibrosis, angiogenesis andinflammation. There is a number of diseases and pathological conditionswherein treatment of an individual in need comprising using suchantibodies and/or peptide fragments, or a pharmaceutical compositioncomprising thereof may lead to successful curing. The term “antibody” inthe present context designates both antibodies, antigen bindingfragments and recombinant proteins thereof.

Thus, the antibodies and/or peptide fragments, or a pharmaceuticalcomposition comprising thereof may successfully be used for the treatingany primary cancer selected from breast-, colorectal-, pancreas-,stomach-, hepatocellular-, other gastrointestinal-, lung-, small celllung-, ovarian-, uterine-, cervix-, testis-, prostate, bladder-, renal-,thyroid- and head/neck carcinoma, malignant melanoma and other skincancers, osteosarcoma, chondrosarcoma, myosarcoma, glioblastoma or otherbrain tumors, germ cell tumors and haematopoietic malignancies.

Or, the antibody and/or peptide fragments, or a pharmaceuticalcomposition comprising thereof may be used for the treating anymetastatic cancer selected from breast-, colorectal-, pancreas-,stomach-, hepatocellular-, other gastrointestinal-, lung-, small celllung-, ovarian-, uterine-, cervix-, testis-, prostate, bladder-, renal-,thyroid- and head/neck carcinoma, malignant melanoma and other skincancers, osteosarcoma, chondrosarcoma, myosarcoma, glioblastoma or otherbrain tumors, germ cell tumors and haematopoietic malignancies.

Yet, in another embodiment, the antibody and/or peptide fragments, or apharmaceutical composition comprising thereof may be used for thetreating an inflammatory disease selected from e.g. rheumatoidarthritis, other inflammatory joint diseases, bacterial infection,active inflammatory bowel disease or liver fibrosis (e.g. caused byhepatitis B or C virus or alcohol abuse).

Still, in other embodiments, the antibody, and/or peptide fragments, ora pharmaceutical composition comprising thereof may also be used for the

-   -   inhibiting proliferation of any non-tumor cells;    -   inhibiting angiogenesis;    -   inhibiting extracellular matrix remodelling;    -   inhibiting of tissue fibrosis,        at any pathological condition, such as for example rheumatoid        arthritis, giant cell arteritis, ankylosing spondylitis,        tuberculosis, sarcoidosis, scleroderma, Crohns disease,        ulcerative colitis, alcoholic liver fibrosis, hepatitis C        wherein said inhibition may be advantageous for curing.

In yet a further aspect the invention relates to a method of treating adisease or condition as discussed above by administering the antibodyand/or peptide fragments of the invention, or a pharmaceuticalcomposition comprising thereof.

Additionally, the invention concerns using antibodies and/or peptidefragments of the invention, or a pharmaceutical composition comprisingthereof in in vitro assays and methods, for example in assays forscreening new anti-cancer compounds.

EXAMPLES 1. Inhibition of Cancer Cell Growth and Survival by Anti-HumanYKL-40 Monoclonal Antibodies In Vitro

Cells of human malignant glioblastoma U87, human osteosarcomas MG63 andU2OS, and human malignant melanoma SK-MEL28 were routinely maintained inculture in Dulbecco's modified Eagle's medium (DMEM) (Gibco),supplemented with 10% foetal calf serum (FCS) and antibiotics(penicillin and streptomycin). All cell lines were obtained from ATTC.

Anti-human YKL-40 antibodies (Ab):

116F9 (mouse monoclonal; highest Ab dilution 1:100 is equal to Abconcentration 0.06 mg/ml115F9 (mouse monoclonal; highest Ab dilution 1:50 is equal to Abconcentration 0.03 mg/ml)201F9 (mouse monoclonal; highest Ab dilution 1:250 is equal to Abconcentration 0.03 mg/ml)R668 (rabbit polyclonal; highest Ab dilution 1:50 is equal to Abconcentration 0.0075 mg/ml)

Procedure:

Cells were seeded in 96-well plates (12 wells×8 rows) with a density of5000 cells/well in 200 μl media. The cells were allowed to attach andgrow overnight. Next day the growth media was discarded and cells wererinsed with phosphate buffered saline (PBS). Subsequently, cells weretreated with 200% fresh media containing different dilutions of theantibodies as shown below:

Plate 1 + 2 Plate 3 + 4 Plate 5 + 6 Plate 7 + 8 Row 115F9 116F9 201F9R667 1 A: Negative Negative Negative Negative 2 B: 1:50 NegativeNegative 1:50 3 C: 1:100 1:100 Negative 1:100 4 D: 1:250 1:250 1:2501:250 5 E: 1:500 1:500 1:500 1:500 6 F: 1:1000 1:1000 1:1000 1:1000 7 G:1:2500 1:2500 1:2500 1:2500 8 H: 1:5000 1:5000 1:5000 1:5000

On each plate wells 1 to 6 of each row were incubated in the presence of10% FCS in the media, wells 7 to 12 were incubated in the presence of0.1% bovine serum albumine (BSA) without FCS in the media.

One plate of each antibody treatment was incubated at normoxiaconditions, and another plate at hypoxia (0.1%) conditions for 72 hoursbefore treatment with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma).

MTT Assay:

MTT-assay (allows to distinguish between living and dead cells in theculture) was performed according to Mossmann T. J Immunol Methods65:55-63, 1983.

40 μl MTT was added to each well (2 mg/ml stock solution) and the plateswere incubated for 1 hour at 37° C. in a normoxic incubator. Afterwardsthe media was discarded and 100 μl DMSO added to each well. Plates wereshaken for 10 sec and the OD (optical density) values were read at 570nm (with the background correction on 690 nm). Results are given as anavarage of OD in 6 wells+/−standard deviation.

As it can been seen from FIG. 1 monoclonal antibodies (MAb) 116F9 and201F9 have a repressive effect on growth of U87 glioblastoma cellsreflected by a reduced number of cells in treated wells (with antibody)compared to control wells (without antibody named “Kontrol” in FIG. 1and “Negative” in FIG. 2) after 72 hours of treatment independently ofthe presence FCS in the culture media. Monoclonal Ab 115F9 andpolyclonal Ab R667 (not shown) did not inhibit the growth of testedcancer cells. MAb116F9 was further tested in cultures of two humanosteosarcoma cell lines MG63 and U2OS and one human malignant melanomacell line SK-MEL-28. The results shown in FIG. 2 demonstrate that MAb116F9 has a growth repressive effect in both osteosarcoma cells, but notin melanoma SK-MEL-28 cells. The two tested osteosarcoma cell lines arecharacterized by a high expression level of YKL-40, whereas SK-MEL-28cells line has a very low expression level, if any.

These results demonstrate that monoclonal antibodies against YKL-40 (MAb116F9 and 201F9) have the growth repressive effect in YKL-40 expressingcancer cells. The effect may be due to inhibition of cell growth andsurvival or induction of apoptosis, or due to both.

Example 2 Inhibition of Cancer Cell Growth by Anti Human YKL-40Monoclonal Antibodies In Vivo

Human glioblastoma U87 cells (4×10⁶ cells/tumor) were injectedsubcutaneous in nude mice. When tumors reached an average volume of 100mm³, the mice were divided in 3 groups of ten mice each. Group A weretreated with 40 mg/kg MAb 201F9 i.p. from treatment day 1 and twice aweek hereafter. Group B were treated with 28 mg/kg MAb 116F9 i.p. fromtreatment day 1 and twice a week hereafter. Group C (control) receivedPBS (phosphate buffered NaCl) i.p. twice weekly. Tumor size was measureddaily and the mice were sacrificed when tumor size reached the volume of1000 mm³. Effect of the treatments on the growth of individual tumorsuntil 600 mm³ is shown in FIG. 3 (Kaplain-Meier curves). It can be seenthat twice-weekly injections of MAb 201F9 (40 mg/kg) reduces growth ofU87 tumors compared to tumors in PBS treated animals (Log rank testp<0.05). This is the case for two different time terms of treatment—thedays until the tumor is reached the volume of 600 mm³ and the days tothe volume of 900 mm³ (not shown). FIG. 3 shows that MAb 116F9 had nosignificant inhibiting effect on growth of U87 tumors compared to growthof the tumors in PBS treated nude mice. However, mice treated with 116F9received a lower dose of the antibody compared to mice treated withantibody 201F9, and one could speculate that 116F9 given at a higherdose might have an inhibiting effect on tumor growth.

In another experiment human glioblastoma U87 cells (8×10⁶ cells/tumor)were injected subcutaneous in nude mice. When tumors reached an averagevolume of 200 mm³, the mice were divided in 4 groups of ten mice each.Group A and C received 8 Gy of ionising radiation (IR) at treatmentday 1. Group A and B were treated with 40 mg/kg MAb 201F9 i.p. fromtreatment day 1 and twice a week hereafter. Group D (control) receivedPBS (phosphate buffered NaCl) i.p. twice weekly. Tumor size was measureddaily and the mice were sacrificed when tumor size reached the volume of1000 mm³. Effect of the treatments on the growth of tumors until 600 mm³is shown in FIG. 4.

Results demonstrated in FIG. 4 show that ionizing radiation (IR) hasalso a significant growth reducing effect on the tumors, which iscomparable to the effect of the antibody MAb 201F). Simultaneoustreatment with MAb 201F9 and IR does not lead to a synergetic effect ofMab and IR. The P-values for log-rank test are summarized in table 1below:

T600 T900 PBS v. MAb P = 0.0465 P = 0.0472 PBS v. IR P = 0.021 P =0.0067 IR v. IR + MAb P = 0.877 P = 0.8235 P—values are Log-rank test.IR—ionizing radiation MAb—monoclonal Ab 201F9 T600—period of time untilthe volume of tumor grown to 600 mm³ T900—period of time until thevolume of tumor grown to 900 mm³

Example 3 YKL-40 Epitope Mapping for mAb 201F9

The epitope mapping was performed by analysing binding of antibody 201F9to peptide fragments of human YKL-40 (Swissprot As. No.: P36222; SEQ IDNO: 1). A peptide library of 381 overlapping peptide fragments (of 8-14amino acid residues) of YKL-40 sequence was synthesized and screened fora capability of binding to 201F9 Ab by Pepscan Systems BV (TheNetherlands)

Several groups of the peptides covering the areas of YKL-40 comprisingamino acid residues 81-105, 127-165, 203-223, 279-292, 300-316 and318-335 demonstrated significant binding. Based on these data andanalysis of structural areas of YKL-40, which include these peptidefragments, a new group of peptides was designed, synthesized andscreened for antibody binding. The new peptides were designedconsidering the position of a peptide sequence in a loop structure ofthe YKL-40 protein to be favourable.

Six peptides covering residues 83-90 (SEQ ID NO: 2), 96-105 (SEQ ID NO:3), 137-150 (SEQ ID NO: 4), 210-220 (SEQ ID NO: 5), 304-314 (SEQ ID NO:6), and 318-329 (SEQ ID NO: 7) were identified as possible antigenicdeterminants/epitopes for antibody 201F9. FIG. 5 demonstrates thelocation of the peptides in the structure of YKL-40. The sequences ofthe peptide fragments were aligned and it appeared that the sequences ofSEQ ID NOs: 2, 3, 4, 5, 6 and 7 share homology. In table 2 (below) thesequences are aligned and homology of amino acid residues is shown inbold indicating the identical amino acid residue and bold underlined—thehomologous amino acid residue.

TABLE 2 GAWRGTTGHHS (SEQ ID NO: 5) YAT K GNQWVGY (SEQ ID NO: 7)GAWRGTTGHHS (SEQ ID NO: 5) RGATVHRTLGQ (SEQ ID NO: 6) GAWRG T TGHHS (SEQID NO: 5) LAWLY P GRRDKQHF (SEQ ID NO: 4) G A WRGT T GHHS (SEQ ID NO: 5)VG G WN FG S QR (SEQ ID NO: 3) GAWRG T TGHHS (SEQ ID NO: 5) LKNRN P NL(SEQ ID NO: 2)

From the table it appears that residues G, A, W, R, T, P and S are mostprobably the residues that determine antigenic properties of thesequences, and sequence GAWRGTTGHHS (SEQ ID NO: 5) is likely thesequence that comprises the major epitope for antibody 201F9.

Amino acid residues located within the sequences corresponding toresidues 210-220 (SEQ ID NO: 5) and 137-150 (SEQ ID NO: 4) of YKL-40have previously been shown involved in YKL-40-ligand/receptor binding(see for example Houston et al. (2003) J Biol Chem 278:30206-30212, andFusetti et al. (2003) J Biol Chem 278:37753-37760). The present datademonstrate that the sequences also comprise antigenic determinates forantibody 201F9. Occupation of these residues by the antibody inhibitsbiological activity of YKL-40 and therefore leads to the inhibition ofgrowth of cancer cells.

1. An antibody, antigen binding fragment or recombinant protein thereof,which is specific for human YKL-40 (SEQ ID NO: 1), said antibody,binding fragment or recombinant protein thereof capable of inhibitinggrowth, differentiation and/or inducing apoptosis of a cell upon bindingto an epitope on YKL-40.
 2. The antibody, antigen binding fragment orrecombinant protein thereof, according to claim 1, wherein saidantibody, antigen binding fragment or recombinant protein, is capable ofspecifically recognizing and binding to an epitope comprising residues83-90, 96-105, 137-150, 210-220, 304-314 and/or 318-329 of the sequenceof human YKL-40 identified as SEQ ID NO:
 1. 3. The antibody, antigenbinding fragment or recombinant protein thereof, according to claim 2,said wherein said antibody, antigen binding fragment or recombinantprotein capable of specifically recognizing and binding to an epitopecomprising residues 210-220 of SEQ ID NO:
 1. 4-5. (canceled)
 6. Theantibody according to claim 1, wherein the antibody is a monoclonalantibody.
 7. (canceled)
 8. The antibody of claim 6, wherein the antibodyis antibody 201F9. 9-12. (canceled)
 13. The antibody, antigen bindingfragment or recombinant protein thereof of claim 1, wherein the cell isa cancer cell and the cancer cell is of any primary cancer or metastaticcancer selected from breast-, colorectal-, pancreas-, stomach-,hepatocellular-, other gastrointestinal-, lung-, small cell lung-,ovarian-, uterine-, cervix-, testis-, prostate, bladder-, renal-,thyroid- and head/neck carcinoma, malignant melanoma, other skincancers, osteosarcoma, chondrosarcoma, myosarcoma, glioblastoma or otherbrain tumors, germ cell tumors and haematopoietic malignancies. 14-15.(canceled)
 16. An epitope comprising of 3 to 8 amino acid residues ofthe sequence of human YKL-40 identified as SEQ ID NO: 1, said amino acidresidues being selected from the amino acid residues 83-90, 96-105,137-150, 210-220, 304-314 and/or 318-329 of the said sequence.
 17. Theepitope according to claim 16, wherein said epitope comprises a sequenceselected from the sequences identified as SEQ ID NOs: 2, 3, 4, 5, 6 or7, or a fragment or a variant of said sequences. 18-26. (canceled)
 27. Apeptide fragment comprising of at least one sequence selected from anyof the sequences identified as SEQ ID NOS: 2-7, or a fragment or avariant of said sequence.
 28. The peptide fragment according to claim27, said peptide fragment is consisting of at least one sequenceselected from any of the sequences identified as SEQ ID NOS: 2-7, or afragment or a variant of said sequence. 29-31. (canceled)
 32. Thepeptide fragment of claim 27, said fragment is consisting of 8 to 15amino acid residues. 33-34. (canceled)
 35. The peptide fragmentaccording to claim 27, said fragment being capable of binding to YKL-40receptor on a binding site involved in stimulating YKL-40 dependent cellgrowth, differentiation and/or cell survival and stimulating orinhibiting YKL-40 dependent cell growth, differentiation and/or cellsurvival. 36-39. (canceled)
 40. The peptide fragment according to claim28, said fragment is consisting of the sequence GAWRGTTGHIS (SEQ ID NO:5). 41-43. (canceled)
 44. A method for inhibiting cell growth,differentiation, and/or cell survival, said method comprising use of anantibody, antigen binding fragment or recombinant protein according toclaim 1 or use of a peptide fragment comprising of at least one sequenceselected from any of the sequences identified as SEQ ID NOS: 2-7, or afragment or a variant of said sequence.
 45. A method for prevention ofprogression and/or treatment of a pathological condition, wherein saidcondition is characterized by the presence of undesirable cell growth,cell differentiation, cell survival, extracellular matrix remodelling,fibrosis, angiogenesis, inflammation, and metastasis comprisingadministering an antibody, antigen binding fragment or recombinantprotein thereof according to claim 1 to an individual in need thereof.46. The method according to claim 45, wherein the pathological conditionis selected from a solid cancer or hematological malignancy,inflammatory disease or disease associated with an increasedextracellular tissue remodeling and/or development of fibrosis.
 47. Themethod according to claim 46, wherein the cancer is a primary cancer ora metastatic cancer selected from any primary cancers or metastaticcancers selected from breast-, colorectal-, pancreas-, stomach-,hepatocellular-, other gastrointestinal-, lung-, small cell lung-,ovarian-, uterine-, cervix-, testis-, prostate, bladder-, renal-,thyroid- and head/neck carcinoma, malignant melanoma, other skincancers, osteosarcoma, chondrosarcoma, myosarcoma, glioblastoma or otherbrain tumors, germ cell tumors and haematopoietic malignancies. 48.(canceled)
 49. The method according to claim 46, wherein theinflammatory disease is selected from rheumatoid arthritis, bacterialinfection, active inflammatory bowel disease, inflammatory diseases,liver fibrosis, organ fibrosis, tissue fibrosis and skin fibrosis.50-68. (canceled)